Table 5 Workflow: from Cytocam assessments to analysis using AVA software.

Device set-up

The Cytocam imaging is set-up and attached to the computer.

CC-tools software started

The CC-tools software embedded in the computer attached to the Cytocam is started and ready to capture microcirculatory video images.

Device ready to record

The Cytocam device directly records the video images on the hard disc of the computer that it is coupled to. The green START button is active and ready to record.

Disposable cap

The imaging probe of the Cytocam device is covered with a sterile plastic disposable cap and this is confirmed in the CC-tools software before start of the capture

Probe placed on the ROI

The imaging probe of the Cytocam device must be placed on the region of interest (ROI). The tip of the imaging probe must make sure to gently touch the preferred tissue surface, avoiding pressure on the microvascular system.

Focus

During the assessment, the focus is adjusted by the CC-tools software. After assessing a sharp image, the imaging probe of the Cytocam device must be stabilized for recording a minimum of 4 s of stable video footage per adjacent location.

Video-images are stored

The assessed video images by Cytocam are captured by the CC-tools software and is set to analyse, store and catalogue the obtained data on hard disk. The data can be exported now to analyse data also in other dedicated software programs.

Automated vascular analysis (AVA) software

The video-clips are exported as AVI files in CC-tools software, and opened in the AVA software to analyse the microcirculatory flow, density and leukocyte parameters. The steps of the semi-automatic analysis are explained in the handout of the software.

Data output parameters

The following output parameters are assessed by the AVA software:

1) TVD, Total Vessel Density: the ratio of the total length of the microvascular vessels and the total area. This parameter shows the morphological density. Unit: mm/mm²

2) PVD, Perfused Vessel Density: the TVD with the correction of the actual perfused vessels, the software calculates only the vessels with sluggish or continuous flow, and discards the vessel with no flow, stop flow and intermittent flow. The PVD shows the functional density, also the measure for the Functional Capillary Density (FCD). Unit: mm/mm²

3) PPV, Percentage of Perfused Vessel: Percentage of the vessel with a continuous or sluggish flow. Unit: %

4) MFI, Microcirculatory Flow Index: the average flow score, the software divides the total area in 4 quadrants, each quadrant is scored by the observer for; 0 no flow, 1 intermittent flow, 2 sluggish flow, 3 continuous flow. The algorithm embedded in the software calculates an average score between 0 and 3.

5) Rolling, non-rolling and total leukocytes: average of the count of white bands generated in the space–time diagram application (Fig. 10), in at least 4 capillary post-capillary venule units. Unit: (Ls)/C-PCV/4secs